Stem and progenitor cells (SPC) reproduce and maintain developmental potential until specific biological signals induce the cells to differentiate into a specific cell type or tissue type. Adult stem and progenitor cells (ASPC) are small populations of SPC that remain in tissues of an organism following birth and are continuously renewed during a lifetime. In vitro colony assays have demonstrated that bone marrow (BM), mobilized peripheral blood (MPB), and umbilical cord blood (UCB), all contain a variety of ASPC. Bone marrow is particularly rich in multipotential ASPC.
ASPC populations that give rise to a lineage are likely to be heterogeneous. Thus, not all stem cells are CD34+. Similarly, while many common lymphoid progenitors are CD7+ and CD3−, some are CD34+ and others are CD34−. In part, such heterogeneity may reflect the fact that cell surface antigen expression, including CD34 expression, can depend on cell cycle or activation as well as developmental potential.
Some studies have suggested that the most primitive human hematopoietic stem cells (HSC) express the CD34 surface marker (i.e., are CD34+), lack obvious lineage commitment markers (designated Lin−), and express low to undetectable levels of other cell surface markers including CD38, CD71, CD45RA, and Thy-1. See, for example, Terstaypen et al. (1991) Blood 77:1218; Landsdorp et al. (1993) J. Exp. Med. 178:787; Cicuttini et al. (1994) Growth Factors 10:127; De Bruyn et al. (1995) Stem Cells 13:281; Di Giusto et al. (1994) Blood 84:421 (1994); Hao et al. (1995) Blood 86:374; Huang et al. (1994) Blood 83:1515; Muench et al. (1994) Blood 83:3170; and Rusten et al. (1994) Blood 84:1471 Conversely, other studies using long-term murine bone marrow transplant models have indicated that CD34lo/− cells contain a hematopoietic stem cell population that is capable of durably generating lymphoid and myeloid lineages following transplantation. See, for example, Osawa et al. (1996) Science 273:242; Morel et al. (1996) Blood 88:629a; and Jones et al. (1996) Blood 88:487, in which a population of small Lin−CD34lo/− ALDH+ cells capable of durably generating lymphoid and myeloid lineages following engraftment was identified.
Some evidence also suggests that ASPC that appear to be committed to a certain cell lineage, such as blood cells, may retain the ability to form additional tissues, such as muscle or nerves, under appropriate conditions. For example, CD133+ mesenchymal stem-like cells generally express more neuronal cell markers than CD34+/CD133− cells (Padovan et al. (2003) Cell Transplantation 12:839). Additionally, SPC become progressively restricted in their developmental potential. Thus, human stem cell populations that express CD45 and CD34, but not CD38, are highly enriched for multipotential (pluripotent) hematopoietic stem and progenitor cells; whereas, cells that express CD45, CD34, and CD38 concomitantly are more restricted developmentally. Similarly, human stem cells that generate endothelial, but not hematopoietic, colonies in vitro generally express CD31, but not CD45 or CD34. Mesenchymal stem cells generally express CD105 and CD135. Chen et al. (2002)Proc. Nail. Acad. Sci. USA. 99:15468; Chen et al. (2003) Immunity 19:525; Pierelli et al. (2001) Leuk. Lymphoma. 42:1195. Morita et al. (2003) Eur. J. Haematol. 71:351.
Aldehyde dehydrogenase (ALDH) is a marker that can be used to enrich APSC. See, U.S. Pat. No. 6,537,807, herein incorporated by reference in its entirety. A fluorescent ALDH reaction product must be used to identify cells via flow cytometry because the marker is not expressed on the cell surface. See, for example, U.S. Pat. No. 6,627,759, herein incorporated by reference in its entirety. Expression of ALDH is elevated significantly in hematopoietic, neural, and potentially other types of ASPC. Cells can be further enriched by gating on low granularity, i.e., side scatter channel) (SSClo) cells. ALDH-positive cells do not co-segregate with CD34. CD34+ cell populations include ALDHbright (ALDHbr) and ALDHdim cells that, respectively, express high and low levels of enzyme (Storms et al. (1999) Proc. Natl. Acad. Sci. USA 96:9118). The ALDHbr cells include virtually all of the stem cells, as evidenced by this cell population's ability to generate multipotential cell colonies in vitro, its ability to reconstitute NOD-SCID mice over a long term, and its ability to rapidly home to the bone marrow in NOD-SCID mice. Conversely, the ALDHdim populations, despite being CD34+ cells, have very limited colony-forming ability, fail to home effectively, and only generate short-term reconstitution in NOD-SCID animals. Thus, ALDH expression can be used to distinguish and isolate functionally active from functionally inactive ASPC CD34+ cells. Heterogeneity in umbilical cord blood (UCB) ALDHbr populations with regard to CD45 and CD31 expression has also been reported (Hess et al. (2003) Blood, 102:383 A).
Technologies for isolating and preparing therapeutically active ASPC from bone marrow (BM) or mobilized peripheral blood (MPB) are particularly useful because the patient only needs minimally invasive procedures for stem cell therapy. Moreover, because ASPC populations are autologous, the grafts will not be subject to rejection. Allogeneic ASPC populations derived from the BM, MPB, or from umbilical cord blood (UCB) of graft donors are also useful. However, to prevent graft rejection, histocompatible donors and immunosuppressive protocols that do not interfere with graft function are needed.
The therapeutic utility of ASPC is well established, and, while not being bound by any mechanism of action or theory, considerable evidence exists that ASPC cell populations can also generate non-hematopoietic tissues in transplant recipients. See, for example, Verfaillie (2002) Trends in Cell Biol. 12:502; Ferrari et al. (1998) Science 279:1528; Gussoni et al. (1999) Nature 401:390-394; Orlic et al. (2001) Nature 410:701-705; Jackson et al. (2001) J. Clin. Invest. 107:1395-1402; Grant et al. (2002) Nat. Med. 8:607-602; Mezey et al. (2001) Science 290:1779-1782; Brazelton et al. (2000) Science 290:1775-1779; Krause et al. (2001) Cell 105:369-377; Petersen et al. (1999) Science 284:1168-1170; Lagasse et al. (2000) Nat. Med. 6:1229-1234; Rehman et al. (2003) Circulation 107:1164-1169. In addition, some evidence suggests that transplanted ASPC induce host stem cells to repair tissues (Verfaillie (2002) Trends in Cell Biol. 12:502). However, because cell populations from stem cell sources contain only a small percentage of ASPC, there is a need for methods of identifying the functional minority versus the non-functional majority. Once these cells are identified, therapies can be improved using custom-engineered grafts that not only contain all necessary cells for a therapeutic result, but also lack potentially dangerous, contaminating cells. Moreover, identification methods allow the useful ASPC to be concentrated, which reduces the amount of material that must be transplanted, thereby reducing tissue damage and toxicity and increasing efficacy. In addition, such selected cells can be used to generate mesenchymal cells that can be used to repair or replace tissues such as nerves, muscles, and endothelium. Stem cells can also be propagated in vitro and expanded into mesenchymal and hematopoietic cell lines to further increase the number of SPC or tissue cells that can be used for transplantation.